The unique infectious cycle adopted by Chlamydia is characterized by temporal regulation of early, middle, and late genes expressed at appropriate times during the developmental cycle. The precise timing of expression for each of these temporal classes of genes relies on distinct mechanisms of transcriptional control. Chlamydia can also be induced to enter an aberrant state in cell culture called persistence, with development halted midway through the developmental cycle. Despite the importance of temporal gene regulation in development of chlamydial disease, knowledge of the molecular mechanisms involved in regulation is limited. Among these temporal transcriptional controls is an early chlamydial protein, EUO, which has been shown to bind to late gene promoters to inhibit transcription. The objective of the proposed research is to identify the regulatory mechanisms imposed by EUO and regulation of EUO itself. The central hypothesis of the proposed study is that EUO binds and represses late gene promoters during early and middle stages of the developmental cycle, and that this repression is alleviated prior to progression into the late developmental stage. Furthermore, during persistence EUO may act to maintain a persistent state of infection by blocking transition into the late developmental stage. The specifi aims of the proposed study are; 1) Determine promoters bound in vivo by EUO ; 2) Identify the mechanism used to alleviate EUO mediated repression; and 3) Determine the role of EUO in persistence and reactivation. In the first aim, analysis of in vivo binding of EUO to late gene promoters during infection will use Chromatin Immunoprecipitation (ChIP) with subsequent sequencing of the DNA to identify EUO binding sites in vivo. In the second aim, there is evidence that EUO protein levels are downregulated to alleviate repression, and this is likely due to inhibition of translation through a small non-coding RNA (sRNA). A regulatory sRNA will be investigated using a sRNA expression library in conjunction with a reporter system fused to euo to assess translational inhibition. In the third aim, persistence is characterized by a block i late gene expression and we propose that regulation of EUO levels is altered, leading to continuous repression of late genes. We will use the methods from Aims 1 and 2 to investigate this. The proposed research is innovative because it will shed light on an important regulator of the chlamydial developmental cycle. This contribution is significant to the mission of NIAID because insight gleaned from these studies may lead to successful new strategies for treating chlamydial infections by targeting master regulators of the developmental cycle, such as EUO.